Elsevier

Sensors and Actuators B: Chemical

Volume 265, 15 July 2018, Pages 115-125
Sensors and Actuators B: Chemical

Scalable fabrication and application of nanoscale IDE-arrays as multi-electrode platform for label-free biosensing

https://doi.org/10.1016/j.snb.2018.02.174Get rights and content

Highlights

  • We describe a fabrication protocol for nanoscale, multichannel, interdigitated electrode arrays in a wafer scale process.

  • We combine nanoimprint lithography and classical photolithography to realize highly-reliable and reproducible sensor arrays.

  • In our process, by firstly replicating the expensive, nano-machined mold, we established a cost-effective and high throughput protocol.

  • We show that our devices are highly reliable from sensor spot to sensor spot and that they can be used as biosensors.

  • We describe the fabrication process in detail, a functional characterization and a first proof-of-concept application of our devices.

Abstract

The continuous progress in the construction of advanced, miniaturized electrodes provides a promising route towards compact and sensitive biological and chemical sensor platforms. We present a combined micro- and nanofabrication process at wafer-scale with nanoimprint lithography and subsequent photolithography for the realization of ultra-small, interdigitated electrode arrays. Several chips of gold nanoelectrode arrays (NEA) in a 4 × 4 configuration designed as interdigitated electrodes (NEA-IDEs) with finger structures measuring 14 μm in length and 600 nm in width with 600 nm spacing were fabricated simultaneously on 4-inch wafers. Our process involved a nanoimprint lithography step, wet-etching, metal evaporation and nano lift-off followed by optical lithography for metal contact lines and passivation layers. The optimized procedure yielded high-quality NEA-IDEs with reliable electrochemical behavior as inferred from voltammetric and impedimetric analysis. The final array allows the control of all 16 NEA-IDEs in parallel, which can be beneficial for multi-analyte detection. In a proof-of-concept assay, to demonstrate the applicability of the NEA-IDEs for biosensing, the nanostructures were modified with short DNA molecules as recognition elements for the detection of hybridization via impedance spectroscopy. Stable impedance signals were found using the redox system ferri-/ferrocyanide. After hybridization with complementary target DNA the sensors showed an enhancement of the charge transfer resistance. Experiments with different target DNA concentrations demonstrated a dynamic detection range of 1–100 nM. The main advantage of these NEA-IDE structures is that they are small enough to be integrated into typical microchannel dimensions of 50–100 μm for miniaturized lab-on-a-chip biosensor devices in future.

Introduction

The most advanced analytical detection technique is mass-spectrometry for the quantification of known materials, the identification of unknown compounds and the elucidation of the structure and chemical properties of different molecules. Additionally, fluorescence or surface-plasmon resonance yield high sensitivities and accuracies for the detection of analyte molecules, but all of them require large, static and expensive instrumentation and are therefore insufficient for most point-of-care applications [[1], [2], [3], [4], [5], [6], [7]]. As an alternative, electrical sensor platforms such as microelectrode-arrays (MEAs) and ion-sensitive field-effect transistors (ISFETs) provide an easy and flexible approach for bio-detection of receptor signaling from living cells and from a variety of biomolecules [[8], [9], [10], [11], [12]]. Especially label-free detection schemes were demonstrated as promising candidates for fast and cost-effective applications, which should overcome the drawbacks of optical detection techniques. However, well-defined interfaces and stable immobilization protocols for the recognition element are necessary to avoid unspecific interactions [[13], [14], [15], [16]]. Progress of label-free sensors was achieved in the field of DNA biosensors over the past few years. This relates to the reusability, the sensitivity enhancement, the understanding of target DNA composition as well as the detection of PCR products or the online tracking of PCR cycles [14,15,[17], [18], [19]]. In recent years, the strong demand for parallel and miniaturized readout tools has led to an increasing interest for the development of miniaturized electrode arrays for bio-detection and continuous readout of biological processes. The recording of impedance spectra offers various advantages such as portable construction, low power consumption during operation, compatibility with routine microfabrication technology and cost-effectiveness [[20], [21], [22], [23], [24], [25]]. Due to their robust sensor characteristics, array-based platforms have attracted significant attention in the biosensor community, which resulted in the miniaturization of the structures and incorporation of nanomaterials combined with nanofabrication techniques [[26], [27], [28], [29]]. The use of nanoscale transducer surfaces generally results in defined sensor signals and a higher efficiency of the bio-signaling processes compared to their microscale counterparts [29]. Nanoscale electrode arrays (NEAs) with optimized design provide a large surface-to-volume ratio. It was earlier demonstrated in various examples that such NEA systems offer a higher sensitivity to biomolecule detection [29,30]. Mainly the closer guidance of the electric field near the sensor surface was demonstrated to enhance the signal-to-noise ratio for impedimetric detection of biomolecules with very low limits-of-detection [31,32]. The realization of nanoscale sensor-arrays for lab-on-a-chip applications is therefore of high technological and scientific interest not only for diagnostic applications but also towards implementing electrical sensor platforms with few molecule detection capabilities as a basis for next generation sequencing technologies [[33], [34], [35], [36], [37], [38], [39], [40], [41]].

With the advantages that NEAs offer for chemo-/bio-analytical applications, growing interest in the development of scalable nanofabrication techniques is imperative [42]. Several approaches for the realization of NEAs were introduced in recent years, which involve bottom up assembly, the use of different nanomaterials such as metal nanoparticles, one-dimensional nanomaterial and 2D materials as well as top-down structuring using ultra-violet (UV) lithography, laser-scribing, e-beam lithography and nanoimprinting techniques [[43], [44], [45], [46], [47]]. While each of these nanofabrication methods presents unique advantages such as high throughput and reproducibility for micro-sized structures (UV-lithography, laser-scribing), elimination of light diffraction and high resolution (e-beam lithography), an easy and scalable approach like nanoimprint lithography for the fabrication of NEAs, which combines the advantages of all three techniques resulting in an homogeneous surface and identical electrical properties, is highly desired for large scale applications [48]. The limitations of nanoimprint lithography are originated in the e-beam fabrication of the master mold, which is susceptible to be damaged by the imprint process. Therefore, a mold replication process is proposed in this manuscript. It is widely accepted that, in the near future, label-free sensor platforms with high sensitivity and identical characteristics will be able to provide a suitable alternative to concurrent methods for the development of clinical assays [36,49]. In order to realize such label-free sensor platforms nanoimprint lithography (NIL) has emerged in recent years as a superior choice over classical nanofabrication techniques for its high throughput, cost-effectiveness and less complicated handling requirements [[50], [51], [52], [53], [54]].

In this report, we demonstrate an optimized NIL process for the fabrication of nanoscale gold IDE-arrays. We realized a platform with high electrical sensitivity and identical sensor characteristics for scalable applications. Our process included first the fabrication of a 4″ NIL mold using e-beam lithography and a replication process for this mold. For sensor fabrication, we used a two-step lithography process. The nanoimprint mold with the NEA-IDEs and microscale contact areas was used to imprint the IDE-arrays at desired locations on a 4″ silicon wafer. In a subsequent step these nanoscale IDE-arrays were contacted by microscale contact lines using a standard optical photolithography process. A simple photolithography mask enabled the deposition of ‘source’ and ‘drain’ contact lines connecting the NEA-IDEs to contact pads on the outer part of a sensor chip. The width and length of each finger of the NEA-IDEs was 600 nm and 14 μm, respectively, with a 600-nm-gap in-between. Each sensor chip measuring 7 × 7 mm2 consisted of a 4 × 4 NEA with 16 interdigitated fingers in each electrode. As control sensors, we also fabricated microscale IDEs (MEA-IDEs) in the same process at the outer perimeter of the wafers using only the photolithography step for these structures. The NEA-IDE arrays were characterized for their electrochemical properties with the redox system ferri-/ferrocyanide using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). They demonstrated an electrochemical behavior typical for gold surfaces. Subsequently, the NEA-IDEs were evaluated for their suitability as impedance-based sensor platform for the label-free detection of DNA hybridization. We chose a robust and simple DNA assay as a proof-of-concept experiment to demonstrate the device performance. Towards this aim, the sensor chips were wire-bonded onto a printed circuit board (PCB), integrated with a fluidic reservoir on-top and connected to a multichannel impedance analyzer [53,[55], [56], [57], [58], [59], [60]].

Section snippets

Reagents and materials

Poly(methyl methacrylate) (PMMA) (950 K, 4% ethyl lactate from Allresist GmbH, Germany) was used for the e-beam lithography process and Technistrip P1316 (MicroChemicals, Germany) was used for its development. 1H,1H,2H,2H-perfluorooctyltrichlorosilane (FOCTS) (Sigma Aldrich, Germany) acted as anti-sticking layer on the NIL molds. Thermal imprint resist mr I-7020R (Microresist Technology, Germany) and lift-off resist (LOR) 3A (MicroChem, USA) were used for the thermal NIL and subsequent nano

Structural characterization

Wafer-scale fabricated NEA-IDEs as well as the assembly of a multi-electrode chip are shown in Fig. 1. Fig. 1a shows a photograph of the silicon wafer with NEA-IDEs, which was fabricated by the combined NIL and photolithography process. A typical chip with 16 NEA-IDEs sharing a common electrical contact in the middle and 16 individual contacts for each device before and after encapsulation is shown in Fig. 1b and c. The chip measured 7 mm by 7 mm and consisted of 4 × 4 NEA-IDEs with a spacing

Conclusions

With our newly developed fabrication process combining nanoimprint lithography and photolithography for nano- and microscale IDE features we demonstrated a scalable fabrication routine for integration of EIS sensor structures into multichannel chips. We produced 60 NEA-IDE array chips on 4″ wafers in parallel. Our protocol yielded sensor devices containing NEA-IDEs with 16 identical, individually-addressable, electrochemical sensing sites. The possibility of defining high-density nanoscale

Acknowledgements

The manuscript was written with contributions of all authors. All authors have given approval to the final version of the manuscript. This research work was supported by the Stiftung Rheinland-Pfalz für Innovation (no. 1082). Vivek Pachauri acknowledges the support from Euroimmun AG for funding of his position. All the authors thank Detlev Cassel, Andreas Pastille and Walid-Madhat Munief for their help in the clean-room processes and with the surface characterizations.

Lotta E. Delle was born in Bielefeld, Germany, in 1987. She received her M.Sc. in Bioelectronics and Nanotechnology from Hasselt University, Belgium, in 2012. From 2012–2015, she was working as a PhD student in the Biomedical Signaling Group of the University of Applied Sciences Kaiserslautern, Germany. She will defend her PhD at the Faculty of Engineering Technology of Hasselt University. Since 2016, she is affiliated to RAM Group DE GmbH, a research and technology company branch of RAM Group

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    Lotta E. Delle was born in Bielefeld, Germany, in 1987. She received her M.Sc. in Bioelectronics and Nanotechnology from Hasselt University, Belgium, in 2012. From 2012–2015, she was working as a PhD student in the Biomedical Signaling Group of the University of Applied Sciences Kaiserslautern, Germany. She will defend her PhD at the Faculty of Engineering Technology of Hasselt University. Since 2016, she is affiliated to RAM Group DE GmbH, a research and technology company branch of RAM Group Singapore in Zweibrücken, Germany, as a team leader for advanced sensor fabrication and characterization. Her research topics focus on micro- and nanoscale chip fabrication with integration of 2D materials for biosensor applications.

    Vivek Pachauri studied Biological sciences (Bachelors – Biology, 2003) and Chemical sciences (Masters- Chemistry, 2005) in Dr. Bhim Rao Ambedkar University Agra, in India. Starting out his research work as a master’s student at the Department of Science and Technology Unit on Nanoscience and Nanotechnology at Indian Institute of Technology Madras, he has focused on chemistry based approaches for the assembly of nanomaterials for electrical and optical applications. In 2006, as a research fellow at the Department of Chemistry, Indian Institute of Technology Delhi, Vivek Pachauri worked on sol-gel chemistry of chalcogenide nanomaterials. He started his doctoral work under the guidance of Prof. Klaus Kern at the Max Planck Institute for Solid State Research Stuttgart and graduated in 2011 from the Swiss Federal Institute of Technology Lausanne (EPFL) with a PhD in Physics. Since his doctoral work, Vivek Pachauri has been constantly focusing on novel pathways towards integration of chemical approaches with large-scale nanofabrication techniques for development of nanoscale-systems based on nanowire, Graphene, 2D semiconductors and nanoparticles for electronic and optical sensor applications. Since 2012 he is a postdoctoral fellow in the Biomedical Signaling Group at the University of Applied Sciences Kaiserslautern, Germany.

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    Marc Riedel was born in Bad Saarow, Germany, in 1988. He received the M.Sc. in Biosystems Technology/Bioinformatics at the Technical University of Applied Sciences Wildau in 2014, scientifically focused on impedance based DNA biosensors. Currently he is finalizing his PhD at the TU Wildau/University of Potsdam in Germany. His research interests include label-free biosensors and light-triggered biohybrid systems for bioelectronics, energy and sensing applications.

    Bert Lägel is a Senior Scientist at the Nano Structuring Center at the University of Kaiserslautern, Germany. He received his PhD degree in materials science from the Robert Gordon University, Aberdeen, UK in 2000 and is now specialized in the field of micro- and nanostructuring techniques, in particular electron beam lithography.

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    Xuan Thang Vu was born in Thai Binh, Vietnam, in 1979. He holds the Bachelor degree in Materials Science from the Hanoi University of Sciences, Vietnam National University, Hanoi and Master degrees in Materials Sciences from the Hanoi University of Sciences and Technology, Vietnam. From 2006–2010 he was working as a PhD student at Institute for Bio-and Nanosystems, Forschungszentrum Jülich, Germany. He received his Dr. rer. nat degree in Physics in 2011 from RWTH-Aachen University, Germany. From 2011, he was working as postdoctoral researcher at University of Applied Sciences Kaiserslautern, Germany. There, he was focusing on developing of integrated systems based on top-down processed silicon nanowire, planar open-gate field-effect transistor, graphene and polymer-based field-effect transistor for DNA, antigen-antibody detections, cell impedance sensing and extracellular action potential recording. Since beginning of 2014, he has joined the phase-change materials research group at Institute of Physics 1A, RWTH-Aachen University, Germany. His current research interest is drift behaviors and switching kinetics of the melt-quenched state of phase-change materials on confined lateral nanoscale line cell.

    Patrick Wagner received a PhD in Physics in 1994 at TU Darmstadt, Germany, and was postdoctoral researcher at KU Leuven, Belgium, until 2001 when he was appointed as a professor for biophysics at Hasselt University, Belgium. In 2014, he returned to KU Leuven as a full professor for bio-functional surfaces and sensors. Patrick Wagner has received several grants and distinctions, including a Marie-Curie Fellowship of the European Union and a Methusalem Fellowship of the Flemish Government. He was president of the Belgian Physical Society in 2006–2007, serves as guest editor of the annual issue ‘Engineering of Functional Interfaces’ in Physica Status Solidi A since 2008, and became editor-in-chief of the newly founded Elsevier journal Physics-in-Medicine in 2016. Regarding novel biosensing technologies, P. Wagner is one of the inventors of the label-free heat-transfer method HTM with a wide variety of bioanalytical applications such as DNA-mutation analysis and cell identification.

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